Prostate cancer remains the most commonly diagnosed cancer and second leading cancer-related death in men in the United States with 240,890 new diagnosis and ~33,720 deaths in 2011 (ACS). Accurate diagnosis and early intervention will reduce the number of deaths due to metastatic prostate cancer. Prostate cancer diagnosis is based initially on the controversial serum prostate specific antigen (PSA) level. To overcome the problems associated with testing PSA level, identification of accurate and reliable cancer-specific molecular markers will reduce unnecessary biopsies for those with benign conditions and direct patients with aggressive disease for appropriate treatment. The genetic basis of prostate cancer can be classified into groups based on specific driving molecular aberrations. About 50-60% of prostate cancer patients are known to have E26 transformation specific (ETS) gene rearrangements and overexpression of SPINK1 was identified in 5-10% of ETS-negative prostate cancer. Recently, we reported the identification of druggable gene fusions involving RAF kinase genes (SLC45A3-BRAF and ESRP1-RAF1) in 1-2% of ETS negative prostate cancer. The genetic basis of the remaining 30-40% of ETS-negative prostate cancer remains unknown. In this proposal we will utilize next generation sequencing technology to study the transcriptional landscape of prostate cancer on a genome-wide scale for pseudogene expression. In our pilot study using 89 prostate cancer samples we identified 8 candidate pseudogenes and a novel fusion gene involving a pseudogene expressed only in prostate cancer. In specific aim 1: We will validate the expression of 8 candidate pseudogenes in a large cohort of prostate cancer and whether each candidate identifies a distinct molecular subtype based on mutually exclusive expression pattern or play a cooperative role in prostate cancer development. In our preliminary validation studies, of the 8 candidate genes we selected CXADR pseudogene (CXADR-pseudogene) because of the tumor suppressor role of the cognate wild-type gene. We confirmed the expression of CXADR- pseudogene in ETS fusion-negative prostate cancer. Further, we unexpectedly identified a fusion gene involving KLK4, a protein coding gene and KRSP1, a non-coding pseudogene expressed only in prostate cancer. Here, we propose to validate these markers in post-DRE urine to develop non-invasive diagnostic/prognostic tools. In specific aim 2: We will examine the functional role of CXADR- pseudogene and KLK4-KRSP1 fusion in in vitro and in vivo experiments. Additionally we will explore the potential for CXADR- pseudogene and KLK4-KRSP1 to serve as biomarkers and we will develop diagnostic reagents based on PCR, immunohistochemistry and RNA in situ hybridization for reliable detection in needle biopsy, prostatectomy and in post-DRE urine samples. We anticipate that validation of these new molecular markers will add into the armamentarium of prostate cancer diagnostics to identify patients that will develop metastatic disease who require early and aggressive treatment strategies.